Process And Device For Determining The Activity Of Enzymes In Liquids, Or The Concentration And/Or Activity Of Inhibitors In Liquids

ABSTRACT

A process and device are disclosed to determine the activity of enzymes in liquids in a largely automatic manner. The device for carrying out this process has a column ( 1 ) with a chromatographic carrier for treating a measurement sample. The carrier is mixed with a substance capable of binding to an enzyme inhibitor present in the measurement sample and that corresponds to at least one enzyme. A measurement sample supply ( 2 ) is associated to one end of the column ( 1 ). A valve/pump arrangement ( 7, 11, 14, 15 ) for filling at least one test tube ( 5 ) with a carrier and at least part of the measurement sample is connected downstream of the column ( 1 ), in the flow direction of the measurement sample. The carrier is dissociated into cleavage products by the action of the enzyme. The rise in concentration per unit of time of at least one of the cleavage products of the carrier is sensed during an incubation time. As an alternative or supplementary step, the enzyme that corresponds to at least one enzyme inhibitor is extracted by chromatography from a measurement sample to detect enzyme inhibitors in liquids and the thus treated measurement sample is tested for inhibitor concentration and/or activity.

The invention discloses a process and a device for determining theactivity of enzymes in liquids, or the concentration and/or activity ofinhibitors in liquids.

The determination of enzyme activities in extracts of plants,suspensions of bacteria, homogenates and body fluids such as bloodserum, blood plasma, urine, punctate, liquor, cell lines or homogenatedtissue has obtained an essential importance for diagnosis, follow up andtherapy control.

For example, the discrimination between enzyme proteins and the otherproteins in blood serum by chemical means is exceedingly questionable,because the concentrations of the individual enzymes in body fluids areextremely small. The concentration of glutamate-oxaloacetatetransaminase in blood serum of a healthy individual is 0.1 μg/ml, forexample. As for comparison, the total protein concentration in bloodserum is in the range between 60 to 80 mg/ml, that is, the ratio ofthese two concentrations is about 1:700.000. Since the determination ofthe enzyme concentration by chemical means is questionable, the activityof the enzyme is calculated from the rate of its reaction with asuitable substrate.

The so called ELISA assay is known from clinical chemistry. Thisimmunoassay measures the concentration of an enzyme and itscorresponding enzyme-inhibitor complex present in a tissue or any othersample. Measuring the enzyme activity by this method is impossible, asit measures concentrations regardless to the actual status of theenzyme, active or inhibited.

According to known techniques the activity of an enzyme is measured insuch cases by removing at first the enzyme inhibitors corresponding tothe enzyme and afterwards determining the activity of the enzyme. Theknown processes are very laborious, referring to the samples to bemeasured being tissues as well as to the method, how to remove theenzyme inhibitors from the sample. This method comprises adding to thesample a substance capable of binding the enzyme inhibitors, incubatingthe so manipulated sample during a definite time to complete the bindingof the enzyme inhibitors along with as much homogenous mixing aspossible of the sample with the said substance and finally, separatingthe so manipulated sample from the said substance.

Starting from this point, the purpose of the present invention is todisclose a process and a device for measuring the activity of enzymes influids by running the method of measurement largely or completelyautomatically and also effectively, and for determining theconcentration and/or activity of inhibitors in fluids additionally oralternatively.

The inventive process manages the above mentioned problem according tothe features of the claims. Accordingly, a process is arranged formeasuring enzyme activities in fluids, which comprises withdrawingenzyme inhibitors, that correspond to at least one of the enzymes in thesample, or enzymes, that correspond at least to one inhibitor, adding asubstrate to the sample manipulated in this manner, so as to getcleavage products from the substrate by reacting with the enzyme, anddetecting the increasing concentration per unit of time of at least oneof these cleavage products during an incubation time. The said processis characterized by withdrawing the enzyme inhibitors or enzymes fromthe sample by means of chromatography.

According to the said invention, it was discovered, that it is possibleto remove the enzyme inhibitors from the sample without as muchhomogenous mixing as possible of the corresponding substance and thesample, and without a following laborious separation process. Inconnection with this, it was discovered, that one can perform mixing andseparation virtually in one step. The said invention has the specialadvantage, that it enables one now to measure enzyme activities in fluidsamples, that is, all kinds of body fluids as well as homogenatedtissues, whereby the withdrawing of the enzyme inhibitors by means ofchromatography makes it possible, to run the process largelyautomatically.

For that purpose, the sample is passed in a useful manner through acolumn filled with a chromatographic carrier, that is treated with asubstance capable of binding the enzyme inhibitors. As a result, theenzyme inhibitor is getting concentrated on the column, so as to achieveat the same time an isolation method for these enzyme inhibitors, in asort of way a side effect of the inventive process.

In order to correlate the results of different measurements, it isnecessary to keep to definite experimental conditions. This can be doneby diluting the manipulated sample, that is the sample released from theenzyme inhibitors, with a suitable column buffer in a definite manner.Moreover, one may admix a suitable measuring buffer to the sampledepending on experimental conditions and the enzyme activities to bemeasured. In a convenient variant of the inventive process, the assay,that is, the mostly diluted sample together with the test substrate,which reacts with the enzyme to be measured to yield cleavage products,is thermostated during the incubation time.

On principle, there are various possibilities, to detect theconcentration increase per unit of time of at least one of the cleavageproducts of the substrate during the incubation time. It is especiallyuseful, to detect the increase of the concentration by means offluorescence measurements, as in-this way one can observe theconcentration increase during the reaction time particularly well.

Moreover, the purpose of the present invention is accomplished by adevice in order to measure the activity of enzymes in fluids accordingto the feature of claim 7. Accordingly, there is provided a columnfilled with a chromatographic carrier, which is treated with a substancecapable of binding enzyme inhibitors corresponding to at least one ofthe enzymes present in the sample. A sample supply tube is connected inseries to one end of the column. To the other end of the column avalve/pump arrangement is connected in series, by which one can fill asubstrate as well as at least a portion of the sample into the testtube. Finally, there is provided a detector for measuring theconcentration increase per unit of time of at least one of the cleavageproducts of the substrate.

According to the said invention, it was discovered, that one can run theprocess of determining enzyme activities and activities and/orconcentrations of inhibitors in fluids, in an arrangement consisting ofdevices connected in series. This process is also characterized in thatby means of chromatography enzyme inhibitors corresponding to an enzymeare withdrawn from the sample. In order to do this, the sample isdirected successively through the different stations of the arrangement,so that one needs not withdraw the sample at any of these stations inorder to manipulate it especially. In particular it is convenient, thatthe measurements are carried out automatically, as the individualcomponents of the arrangement are steered and controlled automatically.

An effective arrangement of the inventive device allows one to use thecolumn repeatedly; that is, one can measure several samples serially.For that purpose the column contains an excess of the substance capableof binding the enzyme inhibitors in the various samples. Definitely, thecapacity of the column is only limited by the amount of excess of thissubstance.

Moreover, it is convenient, that the column is exchangeable. In thiscase a used up column can be replaced by a new one. On the other hand,one can prepare the inventive device for different measurements, thatis, for measuring activities of different enzymes. For in that case onehas to remove different enzyme inhibitors from different samples.Therefore, one has to prepare the column with different substances.

It is convenient with regard to the measuring process runningautomatically, when the sample supply tube can be fed alternatively froma sample supply, such as a rondel, or from a reservoir containing columnbuffer. The column buffer is passed through the column, in order toavoid, that the preceding sample mingle with the following differentsample giving inaccurate values. As long as the process is runningautomatically, the sample supply tube will be fed alternatively from thesample supply or from the reservoir, which is filled with column buffer.In this case it is also convenient, that there is a control deviceprovided, which is connected in series to the column in order to checkthe purity of the column buffer discharged from the column. Such acontrol device may work, for example, by means of photometry or mayinclude means for measuring the electrical conductance of fluidsdischarged from the column.

As already mentioned, the column buffer, which is applied just after asample has passed through the column, serves as a wash liquid for thecolumn and dilutes the sample. In order to evaluate the recorded valuesand to establish definite experimental conditions, in a convenientarrangement of the inventive device, a measuring device is connected inseries to the column to determine the degree of dilution of the samplewith the column buffer. Such a device may include, for example, means ofmeasuring the volume of liquids. Usually, the volume of the sample isknown, therefore, it is sufficient, to determine the volume of the addedcolumn buffer or the total volume of the sample plus the added columnbuffer.

According to the kind of sample and the applied column buffer, it may beconvenient also to provide a device in order to get a homogenous mixtureof the manipulated sample and the column buffer.

Often it is necessary, to mix to the sample, and if need be, to thecolumn buffer and to the substrate in the test tube a measuring bufferby means of the valve/pump arrangement in order to establish definiteexperimental conditions. In this connection means turned out to beconvenient for thermostating the test tube.

As already suggested in connection with the inventive process, it isconvenient, that the detector for detecting the concentration increaseof one of the cleavage products of the substrate includes a fluorescencespectrometer.

For diagnosing it is often necessary to compare measurements of enzymeactivities from untreated with such form treated blood serum. For thatpurpose, it is convenient, that there is provided at least one switchingvalve between the sample supply tube and the column of this inventivedevice, which enables one, to discharge the sample in the test tubealternatively by passing through the column or by bypassing the column.In this way, untreated samples can be measured, as well as such samples,from which the enzyme inhibitors are removed.

In a convenient improved embodiment of the inventive device, it isprovided at least one additional valve in order to feed the column andthe valve/pump arrangement with a buffer, that serves as wash liquid. Insuch a convenient way, it is possible to wash the whole apparatus.

Finally, it may be mentioned, that the inventive device can be runautomatically by means of a computer, which controls the sample-feeding,and if need be, the feeding with column buffer, and coordinates thedetermination of the dilution of the sample, and if need be, the mixingand charging of the test tubes. Moreover, the computer may serve torecord and evaluate the concentration increase per unit of time of oneof the cleavage products.

There are different ways to arrange and improve the conclusions of thepresent invention in a convenient manner. For this purpose, it isreferred to the claims as well as to the following explanations, thatillustrate together with figures, how this invention is put in practice.In connection with these explanations, preferred arrangements andimproved embodiments of the conclusions of the present invention areelucidated.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 illustrates schematically the sequence of the arrangementaccording to this invention.

FIG. 2 is a graph showing the activity of cathepsin B in varioussamples.

FIG. 3 is a graph showing the amount of protein in various samples.

FIG. 4 is a graph showing the activity of cathepsin B in varioussamples.

FIG. 5 is a graph showing the activity of cathepsin H in varioussamples.

FIG. 6 is a graph showing the activity of cathepsin L in varioussamples.

FIG. 7 is a graph showing the amount of protein of two samples per unitof time.

FIG. 8 is a survival graph showing the activity of cathepsin H of twosamples.

FIG. 9 is representation of device for a quick test.

FIG. 1 illustrates schematically the sequence of the arrangementaccording to this invention.

This device as shown in FIG. 1 enables one to determine automaticallyand serially the enzyme activity of various fluid samples, such ashomogenated tissues or all kinds of body fluids.

The device for analysing the sample comprises, according to theinvention, a column 1 filled with a chromatographic carrier treated witha substance capable of binding such enzyme inhibitors, which correspondto at least one enzyme in the sample.

If one wishes to determine, for example, the activity of the enzymecathepsin H in a sample, one may use Sepharose-gel as chromatographiccarrier prepared with papain as to remove the enzyme inhibitorscorresponding to cathepsin H.

A sample supply tube 2 is connected to the upper end of the column 1. 2is in the illustrated device a portion for removing alternatively asample from the sample supply 3 or column buffer from a reservoir 4.

A valve/pump arrangement is connected in series to the lower end of thecolumn 1 to fill—according to the illustrated device—several test tubes5 with a substrate and at least a portion of the sample.

A possible substrate for detecting the activity of cathepsin H in asample is H-Arg-AMC. The cleavage products of this substrate H-Arg-AMCresulting from the reaction with the enzyme cathepsin H are H-Arg andAMC. As the enzyme activity is always proportional to the concentrationof the cleavage products, it is possible to measure the enzyme activityby recording the concentration increase per unit of time of at least oneof the cleavage products during the incubation. With cathepsin H as theenzyme the concentration increase of AMC is recorded. In the realizeddevice as discussed above a fluorescence spectrometer is used as adetector but not characterized further in the only figure shown.

Subsequently, the various components of the device, according to theonly illustrated figure, are explained in detail.

As already mentioned, the various samples are located in the samplesupply 3 which may be a rondel, for example. By means of the portion 2it is possible to withdraw the samples from the sample supplyautomatically and to pass them through a valve 6 to the column 1. Thechromatographic carrier of the column 1 is prepared in such a manner,that there is an excess of a substance, normally one or several enzymes,capable of binding the intended enzyme inhibitors in the sample. As arule, there is a large excess of this substance in order to use thecolumn repeatedly; that is, several samples can pass through the column.Moreover, the column 1 is exchangeable, so that, if the said substanceis used up, the column 1 may replaced by a new one. The shape anddimensions of the column 1 depend on the intended capacity of the column1 as well as on the portions joined to the column 1. For example, thecolumn may have a large cross-section and a corresponding length or asmall cross-section but a larger length.

To check the operating ability of the column 1, one has to measureregularly samples with known enzyme activity values. This is a simpleway to check, whether the substance capable of binding enzyme inhibitorsis used up.

To make sure, that a preceding sample will not mingle with the followingdifferent sample, one may feed the column by turns with the sample andwith a column buffer from the reservoir via the portion 2 and the valve6. This is done after each application of a sample by means of a pump 7,which is connected in series to the column 1.

A photometer as a control device 8 is connected in series to this pump 7and therefore also in series to the column 1, in order to check thepurity of the column buffer discharged from the column and the pump,respectively. Thus one makes sure, that, before the column 1 is fed withanother sample, the liquid in the column is column buffer only.

After the sample had passed through the column, the column 1 is washedwith column buffer as a wash liquid resulting in a diluted sample. Ameasuring device 9 is also connected in series to the column 1 anddetermines the degree of dilution by means of measuring the volume. Asthe original volume of the sample is known, the volume of the columnbuffer used for washing is detected only. The degree of dilution can nowbe calculated from the sum of the volumes of the sample and the columnbuffer.

A device 10 for mixing is connected in series to the measuring device 9so as to homogenate the mixture of the sample and the column buffer.

The resulting homogenous mixture as well as the substrate from asubstrate reservoir 12 and a measuring buffer from an appropriatereservoir 13 can be supplied to the test tubes 5 via a valve 11. Themeasuring buffer serves for establishing definite experimentalconditions. Other solutions can be added alternatively or additionallyto the mixture, for example a special inhibitor-solution serving as atiter for calibrating the concentration. For this the device has to beequipped with additional vessels for the corresponding solutions.Additionally, in the illustrated device a pump 14 and one more valve 15is connected in series to valve 11 for passing on the now existingmixture of sample, column buffer, substrate and measuring buffer.

The entire device described in this paper can be thermostated. Normally,the enzyme inhibitors are removed from the sample at about 4° C. Inparticular, means, which are however not represented in the only figure,are provided to thermostat test tubes 5. The test tubes should at leastbe thermostated during the incubation time. The standard temperature forthis procedure is about 37° C. However, if there are specialexperimental conditions, facilities may be convenient to select variabletemperatures for thermostating. The incubation time, that is the timefor reaction between sample and substrates, depends on the differentenzymes and substrates, and as a rule, it is between 5 and 15 minutes.

It may be mentioned, that the inventive device enables one to carry outredundant measurements by measuring the activity of the same enzyme of asample in different test tubes and also, after an appropriatepreparation of the column, to measure the activities of differentenzymes of the same sample by using different substrates, that is, thetreated sample reacts with different samples. Finally, it is possible,to determine the activity of one enzyme by reacting with differentsubstrates.

The device as shown in FIG. 1 may be used in such a manner, that samplespass alternatively through the column or directly, via the valves 6 and11, pump 14 and valve 15, into the test tubes. Therefore one may measureconcurrently the enzyme activity of samples having passed through thecolumn and samples having not passed through the column. Consequently,one is able to compare measurements of samples in the presence of enzymeinhibitors with measurements of samples, from which the enzymeinhibitors are removed; that is, to compare measurements carried outwith untreated samples with measurements carried out with treatedsamples.

Additionally, there is also a valve 16 provided in the device shown inFIG. 1 in order to feed the entire apparatus with buffer 17 serving as awash liquid.

By means of a computer 18 one may control the individual components ofthe inventive device, for example, the sample supply portion so as tofeed the column with samples and column buffer. Moreover, the evaluationof the recorded measurements may be done by means of the computer 18.

To sum up, by means of the inventive process and device an efficient andlargely automatical method for determining enzyme activities of fluidsamples can be realized.

As a supplement, a process and a device is proposed for determining theactivity of enzymes and/or the concentration of inhibitors both presentin fluids.

This method may be used in addition or alternatively to the aboveexplained method.

The determination of enzyme activities is very important for diagnosisin clinical chemistry and microbiology as well as in biochemicalresearch. A lot of methods for such determinations are known and used asstandard methods.

However, it has been impossible, to measure the activity of variousenzymes in blood serum, other body fluids, or in cells, where they areproduced, quickly and with a reasonable price, because many of them arepartly or totally inhibited in order to protect their neighborhood; thatis, where they could cause damage, they are mostly inactive.

If a pathological change arises in cells producing such enzymes, or intheir neighborhood, the ratio of inhibited to active amount of enzymemay be changed in favor to the non inhibited, active form; especially inthe blood serum, a resulting excess of enzyme is immediately reversiblyblocked by inhibitors.

Therefore, the free activity of not inhibited enzymes in blood serum,for example, could only be measured by means of hitherto appliedmethods, if the concentration of the enzymes was extremely high.

However, it may be necessary for an early diagnosis of diseases, tomeasure reliably and as quickly as possible the increase of the totalenzyme activity per unit of volume or the ratio of the activities perunit of volume of the inhibited to the free form, for the peakactivities of the free portion of the enzyme as detectable byconventional methods often does not arises before an advanced status ofthe disease.

Moreover, to know the portion of the active form related to the totalpool of the enzyme may also be important in research.

By means of immunoassays (ELISA) the concentration of even the inhibitedportion of a class of enzymes can be determined. However, a considerabledisadvantage of immunoassays is the impossibility to discriminatebetween the free and therefore active and the inhibited state of theenzyme, because these methods detect only the sum of both states of theenzyme together with enzymes, which have lost their catalytic functionpartly and totally. The measurement of activities by means of thesemethods is impossible.

Likewise, the known enzyme assays for measuring the enzyme activity cannot discriminate between both states of the enzyme, as the free statecan merely be measured by these methods.

How can the total activity of an enzyme present in both states,inhibited and free, be measured additionally to the activity of the freestate?

The solution of this problem is simple and has a reasonable price.

By means of chromatography the specific inhibitors inhibiting theintended enzyme in its activity are removed from the sample, which maybe human blood serum. Afterwards a conventional measurement of enzymeactivity can be carried out.

By measuring the activity of the enzyme in the untreated sample, theportion of the free and the inhibited enzyme of the totally presentamount of enzyme can be determined.

The following example may illustrate this process:

Papain, a cysteine protease, is bound to a Sepharose-gel, which servesas a carrier. The gel prepared in this way is filled in a column forchromatography. From a sample, such as homogenated tissue from a tumorof the lungs, incubated in-this column, all inhibitors are removed, thatshow a higher affinity to papain than to the enzymes, to which they areoriginally bound. Such enzymes may be among others cathepsin B, H and L.One of them, cathepsin H, normally is almost completely inhibited andits activity is only measurable after removing the inhibitors (stefin A,kininogen and others).

The measurement of the activities is carried out according to knownassays by means of measuring fluorescence, using substrates andinhibitors suitable for the enzyme.

This process is feasible, according to the application, with the mostdifferent enzymes, inhibitors and materials for chromatography. It maybe carried out completely automatically and therefore, it is qualifiedfor routine measurements in the laboratory (for this see FIG. 1).

A large quantity of samples can so be stored in a sample supply, whichmay be a rondel or a suitable depot for samples. Thereof a definitevolume of the sample can be directed occasionally in a suitablechromatographic column (1) via a device for discharging (2) by means ofa pump connected in series to this portion (2).

After a fixed incubation time, this sample, from which the inhibitorsare now removed, is discharged completely from the column andsimultaneously diluted by passing a definite buffer solution through thecolumn from a reservoir (4) of this buffer via the same way as describedabove for the sample.

A following measurement of the purity (8), by means of photometry ormeasurement of the electrical conductance, for example, enables one toprevent, that some of the sample remains on the column and thatconsumption of buffer is limited to the necessary amount.

By means of an additional device (9) the total volume discharged fromthe column can be measured.

A device (10) is connected in series to the column, to obtain ahomogenous mixture of the sample and the buffer after being dischargedfrom the column.

By means of an additional pump and via a valve, measuring buffer,substrate, the sample, which is diluted in a definite ratio with theinitially used buffer solution, and, if need be, inhibitors, can bedirected in a definite sequence to a device (5) for measuring thefluorescence. The said valve and all other functional units of theapparatus can be controlled by a computer (18).

The activity per unit of volume of the intended enzyme in the originalsample as well as the activity per unit of mass, if the proteinconcentration is known, can now be calculated in a simple way by meansof the computer.

To compare the enzyme activity, determined in the said manner, with theenzyme activity in the untreated sample, one can direct the samplethrough the apparatus without passing through the column.

After or even during the measuring process one can start a computercontrolled flushing of the device with a wash liquid, in order to removecompletely any residue of the sample.

There are additional explanations of FIG. 1 in the parent application.

To solve special problems in the best way by means of the describeddevice, the arrangement of some units, in particular the valves, candiffer from the arrangement shown in FIG. 1.

To improve the efficiency of the said device, it is convenient havingseveral columns connected in series or parallel. Due to providedfacilities for measuring the activity of different enzymes of the samesample in a parallel manner by means of fluorescence measurements; theefficiency of this device is also improved.

Instead of measuring the activity of specific enzymes by means offluorescence measurements, one may carry out this determination by meansof other methods such as photometry, according to need and the appliedassay.

As a special item, one may take columns, repeatedly useable, andprepared with different substances such as different enzymes or enzymefragments produced in a large technical scale, and one may also takechromatographic carriers linked with inhibitors.

Due to these numerous facilities, enzymes as well as inhibitors can bepurified according to the applied column.

If a column prepared with enzymes is used up, the inhibitors removedfrom the samples can be separated from the enzymes by means of a simplechemical procedure and can be put to further use. In this way hithertounknown inhibitors may be concentrated and characterized.

If a column prepared with inhibitors is applied, enzymes may be removedselectively from the sample and may be characterized. In an analogousmanner, as described in the foregoing example, inhibitors, for examplekinogen, are now removed instead of enzymes (such as cathepsin H). Theseinhibitors are analysed by means of specific assays in order todetermine the concentration and activity of them in the original sample.

The figures explain the improved embodiment.

The efficiency of the above described column (the enzyme papain iscoupled to Sepharose-gel, the gel serves as a chromatographic carrier)is demonstrated as an example by means of the following measurementscarried out in connection with research on the proteinases cathepsin B,H and L:

1. In homogenated lung tissues of patients with tumors the activities(μU/mg protein) of cathepsin B, H and L are measured. This is donebefore and after removing the corresponding inhibitors by means ofSepharose-gel prepared with papain. The samples are incubated in thecolumn for fixed 15 min.

FIG. 2 demonstrates, that the enzyme activity of tissue from lungs witha tumor as well as without a tumor is, according to the median values,substantially higher after removing the inhibitors by means of a columnthan in untreated samples.

In this graph, for every comparison of samples before and after removingthe inhibitors, the same collective of samples is used. The increase ofactivities can only be interpreted through removing the inhibitors andgoes along with the standard literature.

2. FIG. 3 illustrates, that the amount of protein (mg/ml) according tothe mean values is substantially higher before the withdrawing of theinhibitors by the papain column than afterwards. This can only beinterpreted through the fact, that inhibitors are really removed.

3. As an example for demonstrating the time of withdrawing of inhibitorby papain, which is coupled to the column, a pair of samples was chosenby chance. The activities were determined after different times ofincubation of the samples on the column.

FIG. 4 illustrates a rapid activity increase of cathepsin B, justbeginning at the starting point and reaching a plateau after 15 minutes.The starting point (t=0 min) corresponds to the activity value beforethe removal of inhibitors.

FIGS. 5 and 6 illustrate also a large activity increase after shortincubation times. The remaining activity resulting from the activitymeasurement of cathepsin L, as indicated in FIG. 6, is the activity ofan enzyme not yet characterized.

4. FIG. 7 demonstrates the course of decrease of the amount of proteinrelated to the sample described in the foregoing paragraph 3. Alreadyafter a short time the amount of protein reaches a minimum and proceedsin the following time to a plateau; this means, that, due to the excessof papain in the column, the inhibitors will be quickly and reliablybound.

5. The survival-graph (Kaplan-Meier-graph) in FIG. 8 demonstrates thefollowing fact: the prognosis for survival-time of patients with acathepsin H activity in the tissue of the tumor, that is beyond athreshold of 533 μEU/mg, is by far more unfavorable than with anactivity below this threshold. The values in this and in all other abovedescribed figures originated from measurements carried out with apapain-column.

The following conclusions, for example, are to be drawn from the abovedescribed explanations:

In many body fluids, such as blood serum, urine or liquor as well as inhomogenated tissues, suspensions of bacteria and other fluids, there areenzymes, which are inactivated by specific inhibitors. Up to now it wasonly possible in case of peak values, to determine the activity of theseenzymes.

By means of immunoassays, such as the comparatively expensive ELISAassay, only the total amount of enzyme concentration can be measured,but not the activity of intact and therefore catalytically active formsof the enzyme.

It is now possible, by means of an appropriately preparedchromatographic column, to remove specific inhibitors from the sample inorder to measure fractions and the total amount of the activity of oneor several enzymes.

A side effect of this method is the purification and a concentration ofspecific inhibitors on the column. In a following step they can beseparated from the chromatographic material in order to characterizethem.

It is also possible, to bind inhibitors, producible in a large technicalscale, instead of enzymes or enzyme fragments, which may also beproduced in a large technical scale, to the chromatographic material, soas to characterize unknown enzymes as well as to determine theconcentration and activity of the inhibitors present in this sample.

Due to the automation, the above described device or a requiredmodification of it, can be used for routine measurements inclinical-chemical laboratories as well as in microbiological diagnosisand in biochemical research.

The chromatographic columns can be used several times and are of lowproduction costs, therefore, it is possible to analyse in a short time alarge number of samples with a reasonable price.

By establishing such a device, a field of research is disclosed,hitherto not treated due to a lack of appropriate techniques.

Due to the described process and device, it is possible, to make aprognosis of the course of a disease and of the survival time of thepatient, in order to set a therapy adapted to the special requirementsof the patient. It will be the task of research, to evaluate by means ofthe said process and device new and reliable tumor markers andparameters for checking the course of a disease.

The said process and device may also be significant for bacteriology andmicrobiology, because the biochemical classification of bacteria, forexample, is of considerable significance for diagnosis.

The above described device is transportable only to a certain extend andnot well adapted to carry out a quick test outside of a laboratory witha reasonable price. Therefore, a device, as described in the followingsection and based on the device illustrated in FIG. 1, is convenient forsuch a quick test.

FIG. 9 illustrates the improved embodiment.

A special syringe (1) represented schematically in states A, B and C,contains in the lower part, between two microfilters (3 and a portion of5), a chromatographic material, to which there are coupled, according tothe above described process, enzymes, inhibitors or fragments thereof,which are produced in a large technical scala.

In the state before use, (A), there is a liquid, appropriate to therequirements, for example a wash liquid, above the chromatographicmaterial and below the plunger (2) in a space (6) filled with liquid,

To reach the state for reaction, (B), a sample, such as blood, bloodserum, urine, liquor or another fluid, is sucked through the aperture(5) of the syringe, which is equipped with a microfilter, in the space,which contains the chromatographic material, by means of a vacuum,produced by the plunger (2). Subsequently, when the occasion arises,there is a definite incubation time. In the state for reaction,according to the chromatographic material, enzymes or, in the case ofpapain bound to Sepharose-gel, inhibitors such as kininogens, areremoved from the sample.

After that, the so treated sample together with the liquid, which is instate A above the filter and below the plunger (2), is discharged in areaction vessel (7) by means of a pressure produced by the plunger (C).

The reaction vessel may contain a specific substrate, which is, forexample, specifically cleaved by enzymes, in order to get a colorchange, like an indicator. These enzymes have just been released fromtheir inhibitors by the foregoing procedure.

After a definite time, the result of this quick test can be recorded, bymeans of the color change in the reaction vessel, for example.

Due to this process, it is possible without a bulky laboratoryapparatus, that is based on the device illustrated in FIG. 1, to douseful diagnosing and prognosing, although the efficiency and precisionof a laboratory apparatus is not achieved. Such a quick test may becarried out to do diagnosing in clinical and preclinical emergency casesas well as in general practice.

1-123. (canceled)
 124. A system for measuring activity of an enzyme in aliquid sample that includes at least one enzyme and at least one enzymeinhibitor corresponding to said enzyme, the system comprising: a testvessel containing said sample; a chromatographic carrier having asubstance capable of binding said enzyme inhibitors corresponding tosaid enzyme in a sample, the substance bound to the carrier effectivefor binding said enzyme inhibitor with a higher binding affinity thansaid enzyme, said chromatographic carrier being capable of contactingsaid sample; and a means for measuring the activity of said enzyme,wherein the means for measuring the activity of said enzyme and saidtest vessel are independently thermostated.
 125. The system according toclaim 124 wherein the chromatographic carrier contains an excess amountof substance capable of binding said enzyme inhibitor relative to theamount of inhibitor in a sample such that the chromatographic carriercontaining the substance capable of binding said enzyme inhibitor can beused repeatedly.
 126. The system according to claim 124 wherein themeans for measuring activity is a fluorescence detector.
 127. The systemaccording to claim 124 wherein the substance bound to the carriereffective for binding an enzyme inhibitor is papain.
 128. A method formeasuring activity of an enzyme in a liquid sample that includes atleast one enzyme and at least one enzyme inhibitor corresponding to saidenzyme, the method comprising: providing said sample to a test vessel;contacting a said sample with a chromatographic carrier having asubstance capable of binding said enzyme inhibitors corresponding tosaid enzyme in a sample, the substance bound to the carrier effectivefor binding said enzyme inhibitor with a higher binding affinity thansaid enzyme; separating said chromatographic carrier having a substancecapable of binding said enzyme inhibitor from said sample; and measuringthe activity of said enzyme with a means for measuring enzyme activity,wherein the means for measuring the activity of said enzyme and saidtest vessel are independently thermostated.
 129. The method according toclaim 128 wherein the chromatographic carrier contains an excess amountof substance capable of binding said enzyme inhibitor relative to theamount of inhibitor in a sample such that the chromatographic carriercontaining the substance capable of binding said enzyme inhibitor can beused repeatedly.
 130. The method according to claim 128 wherein themeans for measuring activity is a fluorescence detector.
 131. The methodaccording to claim 128 wherein the substance bound to the carriereffective for binding an enzyme inhibitor is papain.